1. Field of the Invention
The present invention relates to the field of cellular biology, particularly to the field of cell implantation therapy, more particularly to the development of a source of cells that may be used directly or for the cloning of cells for use in cell implantation therapy, and more particularly to the in vivo dedifferentiation of certain skin cells as implantable cells for the production of cells for transplantation or implantation cell therapies.
2. Background of the Art
Cell implantation requires significant levels of control in the introduction of cells. The cells must be capable of performing the specific functional replacement or addition therapy desired in the transplantation or implantation, and the cells must be compatible with the receiving organism. Present techniques emphasize the use of same species differentiated cells or the use of compatible undifferentiated cells for use in the implantation therapy. Conservative protocol has generally motivated investigators to prefer same species undifferentiated cells or specialized same species differentiated cells for use in such procedures. This considered preference has limited the source of cells that can be used for implantation therapy. For example, viable, mature, differentiated cells are not readily available, and the moral implications of fetal tissue collection of undifferentiated cells has limited the sources of such cells, as well as the natural quantity limitation on the source itself.
Cell implantation using tissue-derived nuclear transfer embryos has been performed, with attendant nuclei replacement to provide the targeted specialty cells for implantation. Bovine skin fibroblast cells were transferred into enucleated bovine oocytes, resulting in a fetus. The ventral mesoncephalon was isolated from the bovine fetus. When transplanted, this tissue successfully ameliorated symptoms in the Parkonsonian rat (Zawada et al., 1998, Nature Medicine, 4:569-574). Skin fibroblast cells from cows, sheep, pigs, monkeys, and rats have been used as sources of donor nuclei, as well as nuclei from human sources.
The clinical management of numerous neurological disorders has been frustrated by the progressive nature of degenerative, traumatic or destructive neurological diseases and the limited efficacy and the serious side-effects of available pharmacological agents. Because many such diseases involve destruction of specific “neuronal clusters” or brain regions, it has been hoped that grafting of neural cells or neuron-like cells directly into the affected brain region might provide therapeutic benefit. Cell transplant approaches have taken on a major emphasis in current Parkinson's disease research, and may prove useful in promoting recovery from other debilitating diseases of the nervous system including Huntington's disease, Alzheimer's disease, severe seizure disorders including epilepsy, familial dysautonomia, as well as injury or trauma to the nervous system. In addition, the characterization of factors which influence neurotransmitter phenotypic expression in cells placed into the brain may lead to a better understanding of normal processes and indicate means by which birth defects resulting from aberrant phenotypic expression can be therapeutically prevented or corrected. Neurons or neuronal-like cells can be grafted into the central nervous system (CNS), in particular, into the brain, either as solid tissue blocks or as dispersed cells. However, to date, a number of problems of both a technical and ethical nature have plagued the development of clinically feasible grafting procedures.
Parkinson's disease results from a selective loss of dopaminergic nigrostriatal neurons, resulting in a loss of input from the substantia nigra to the striatum. Solid grafts of tissues potentially capable of producing dopamine, such as adult adrenal medulla and embryonic substantia nigra (SN), have been used extensively for experimental grafting in rats and primates treated with 6-hydroxydopamine (6-OHDA) to destroy dopaminergic cells (Dunnett, S. B. et al., Brain Res. 215: 147-161 (1981); ibid. 229: 457-470 (1981); Morisha, J. M. et al., Exp. Neurol. 84: 643-654 (1984); Perlow, M. J. et al., Science 204: 643-647 (1979)). Grafts of embryonic SN have also been used as therapy for primates lesioned with the neurotoxin 1-methyl-4-phenyl-1,2,3,4-tetrahydropyridine (MPTP), which produces a Parkinson's-like disease (Redmond, D. E. et al., Lancet 8490: 112-27 (1986)).
The methods of the present invention are useful for treating a number of human neurological disease. Parkinson's Disease can be treated according to the present invention by implanting dopamine-producing cells in the recipient's striatum. Alzheimer's disease involves a deficit in cholinergic cells in the nucleus basalis. Thus, according to the invention, a subject having Alzheimer's disease or at risk therefore may be implanted with cells producing acetylcholine.
Huntington's disease involves a gross wasting of the head of the caudate nucleus and putamen, usually accompanied by moderate disease of the gyrus. A subject suffering from Huntington's disease can be treated by implanting cells producing the neurotransmitters gamma amino butyric acid (GABA), acetylcholine, or a mixture thereof. According to the present invention, the support matrix material to which such cells are attached is preferably implanted into the caudate and putamen.
Epilepsy is not truly a single disease but rather is a symptom produced by an underlying abnormality. One skilled in the art will appreciate that each epileptic subject will have damage or epileptic foci which are unique for the individual. Such foci can be localized using a combination of diagnostic methods well-known in the art, including electroencephalography, computerized axial tomography and magnetic resonance imaging. A patient suffering from epilepsy can be treated according to the present invention by implanting the support matrix material to which GABA-producing cells are attached into the affected site. Since blockers of glutamate receptors and NMDA receptors in the brain have been used to control experimental epilepsy, cells producing molecules which block excitatory amino acid pathways may be used according to the invention. Thus implantation of cells which have been modified as described herein to produce polyamines, such as spermidine, in larger than normal quantities may be useful for treating epilepsy.
The methods of the present invention are intended for use with any mammal that may experience the beneficial effects of the methods of the invention. Foremost among such mammals are humans, although the invention is not intended to be so limited, and is also applicable to veterinary uses.
Thus, while the feasibility of the transplant approach has been established experimentally, this approach is severely limited by the need for the use of fetal tissue or specifically differentiated cells from the same organ of the organism, which is of limited availability and potentially of great political consequence. In essence, transplantation of human fetal tissue from aborted pregnancies has been prohibitive in the United States. It would thus be of great benefit if simple, routine and safe methods for the successful transplantation of socially acceptable and available tissue into the brain were available for the treatment of debilitating neurological disease.
One potential approach to this problem has been the implantation of adult cells, attempted by Aebischer and his colleagues, who have successfully implanted into the brain selectively permeable biocompatible polymer capsules encapsulating fragments of neural tissue which appeared to survive in this environment (Aebischer, P. et al., Brain Res. 448: 364-368 (1988); Winn, S. R. et al., J. Biomed Mater Res. 23: 31-44 (1989). The polymer capsules, consisting of a permselective polyvinyl chloride acrylic copolymer XM-50, completely prevented the invasion of the encapsulated tissue by host cells. Based on the permeability, antibodies and viruses would be expected to be excluded as well. When dopamine-releasing polymer rods were encapsulated into such a permselective polymer and implanted into denervated striatum in rats, alleviation of experimentally-induced Parkinson disease symptoms was achieved (Winn S. R. et al., Exp. Neurol. 105: 244-50 (1989). Furthermore, U.S. Pat. No. 4,892,538 (Aebischer et al., issued Jan. 9, 1990) discloses a cell culture device for implantation in a subject for delivery of a neurotransmitter comprising secreting cells within a semipermeable membrane that permits diffusion of the neurotransmitter while excluding viruses, antibodies and other detrimental agents present in the external environment. The semipermeable membrane is of an acrylic copolymer, polyvinylidene fluoride, polyurethane, polyalginate, cellulose acetal, polysulphone, polyvinyl alcohol, polyacrylonitrile, or their derivatives or mixtures and permits diffusion of solute of up to 50 kD molecular weight. This device was said to be useful in treatment of neurotransmitter-deficient conditions, such as Parkinson's disease, by sustained, local delivery of neurotransmitters, precursors, agonists, fragments, etc., to a target area, especially the brain. The device may be made retrievable so that the contents may be renewed or supplemented, and the cells are protected against immunological response and viral infection.
By the term “neural or paraneural origin” is intended a cell which is derived from the embryonic neural crest. A preferred example of a cell of paraneural origin is an adrenal medullary chromaffin cell. The precursor cells to the mammalian adrenal medulla are of neural crest origin and possess the potential to develop along either neuronal or endocrine lines of differentiation (Bohn, M. C. et al., 1981, supra, Devel. Biol. 89: 299-308 (1982); Unsicker, K., Develop. Biol. 108: 259-268 (1985)). Chromaffin cells from the rat, monkey, and human adrenal medulla, when removed from adrenal cortical influences and exposed to nerve growth factor (NGF), change from an endocrine to a neuronal phenotype (Notter, M. F. et al., Cell Tiss. Res. 244: 69-70 (1986); Stromberg, I. et al., Exp. Brain Res. 60: 335-349 (1985); Unsicker, K. et al., 1978, supra). When co-grafted with cerebral cortical or hippocampal tissue into the anterior chamber of the rat eye, adrenal chromaffin cells form nerve fibers which innervate the adjacent co-grafted brain tissue (Olson, L. A. et al., Exp. Neurol. 70414-426 (1980)). Another paraneural cell type is a retinal pigment epithelium cell (Song, M-K et al., J. Cell. Physiol. 148: 196-203 (1990)).
U.S. Pat. No. 5,958,767 discloses that clones of human NSCs (neural stem cells)—unambiguously affirmed by the presence of a common retroviral insertion site and propagated by either epigenetic or genetic means—can participate in normal CNS development in vivo and respond to normal microenvironmental cues, including migration from various germinal zones along well-established migratory routes to widely disseminated regions. A single NSC is capable of giving rise to progeny in all 3 fundamental neural lineages—neurons (of various types), oligodendroglia, and astroglia (hence, multipotency)—as well as giving rise to new NSCs with similar potential (i.e., self-renewal). In vivo, following transplantation into mouse hosts, a given human NSC clone is sufficiently plastic to differentiate into neural cells of region- and developmental stage-appropriate lineages along the length of the neural axis: into neurons where neurogenesis normally persists, and into glia where gliogenesis predominates, emulating patterns well-established for endogenous murine progenitors, with which they intermingle seamlessly. Thus, for example, they will give rise to neurons following migration into the Olfactory Bulb (OB) at one end of the neuraxis and into granule neurons in the cerebellum at the other, yet also yield astroglia and oligodendroglia, the appropriate cell types born in the postnatal neocortex, subcortical white matter, and striatum. Of additional significance, as might be expected of a true stem cell, many of the neuronal types into which these NSCs could differentiate, are born not at the developmental stage from which the cells were initially obtained (e.g. midgestation), but rather at the stage and region of NSC implantation, thus affirming appropriate temporal (in addition to regional) developmental responsiveness.
Neural stem cells were thought to have limited differentiation capabilities within the species. However, adult mouse NSC were transferred into early mouse and chick embryos and contributed to all three germ layers in the developing mouse and chick embryo. This demonstrated that these particular NSC cells were capable of differentiating into multiple cell types (Clarke et al., 2000 Science vol 288:1660). The mechanism was not stated to be effective for other than the chosen organisms, there was no specific mechanism described such as transdifferentiaion or mutation, and there was no indication that determined cells could actually reverse their direction of differentiating and differentiate in a different direction in human neural stems cells, which are quite advanced compared to the cells selected in this analysis.
Stenevi et al. (Brain Res. 114: 1-20 (1976) found that the best results were obtained with fetal CNS neurons which were placed next to a rich vascular supply. In fact, a review of the literature reveals that tissue from almost every area of the fetal brain can be successfully transplanted if care is taken with procedural details (see, for example, Olson, L. A. et al., In: Neural Transplants: Development and Function, Sladek, J. R. et al., eds,. Plenum Press. New York, 1984, pp. 125-165).
Embryonic tissue provides an excellent source of cells that will differentiate in a foreign environment and become integrated with the host tissue. For example, grafts of embryonic SN into 6-OHDA treated rats have been shown to produce dopamine, to reduce apomorphine- or amphetamine-induced rotation, to alleviate sensory deficits and to make synapses in the host striatum (Dunnett et al., Morisha et al., Perlow et al., supra). Grafted neurons are also spontaneously active, thus mimicking normal adult SN neurons (Wuerthele, S. M. et al., In: Catecholamines, Part B, (E. Usdin et al., eds.), A. R. Liss, Inc., New York, pp. 333-341).
In contrast to successful grafting of fetal neural tissue, mature CNS neurons have never been found to survive in transplants (Stenevi, U. et al., Brain Res. 114: 1-20 (1976)). The reason fetal CNS neurons survive grafting procedures, while adult neurons do not is uncertain, but probably related to several factors. First, fetal neurons are less affected by low oxygen levels than mature neurons (Jilek, L., In: Developmental Neurobiology, Himwich, W. A., ed., C. C. Thomas Publisher, Springfield, Ill., 1970, pp. 331-369), and grafting procedures necessarily involve periods of anoxia until an adequate blood supply to the transplant is established. Secondly, fetal neurons seem to survive best when they are taken during a rapid growth phase and before connections are established with target tissues (Boer, G. J. et al., Neuroscience 15: 1087-1109, (1985)). Also, fetal tissue may be especially responsive to growth (or “survival”) factors that are known to be present in the milieu of the damaged host brain (Nieto-Sampedro, M. et al., Science 217: 860-861 (1982); Proc. Natl. Acad. Sci. USA 81: 6250-6254 (1984)).
In further human studies (Lieberman, supra; Lindvall, O., J. Neurol. Neurosurg. Psychiat., 1989, Special Supplement, pp. 39-54; Bakay, R. A. E., Neurosurg. Clin. N. Amer. 1: 881-895 (1990)), autologous grafts have been attempted to replace the need for fetal material. In this procedure the patients first underwent initial abdominal surgery for the removal of a healthy adrenal gland. The patient then was subjected to similar neurosurgery as that for the fetal adrenal transplant. The surgical morbidity-mortality for the combined adrenalectomy/neurosurgery was expectedly high. The ultimate therapeutic result was claimed to be as high as 30% but may have been as low as one patient in the series of six. There was no evidence that the adrenal material transplanted into these patients survived.
However, despite the promise of fetal tissue and cell transplants, the art has turned to alternate sources of donor tissues for transplantation because of the ethical, moral, and legal problems attendant to utilizing fetal tissue in human medicine. These sources include neural and paraneural cells from organ donors and cultured cell lines. (See, for example: Gash, D. M. et al., In: Neural Grafting in the Mammalian CNS, Bjorklund, A. et al., eds, Elsevier, Amsterdam, 1985, pp. 595-603; Gash, D. M. et al., Science 233: 1420-22 (1986)).
There are suggestions in the literature that there may be an additional advantage of grafting dissociated cells compared to blocks of tissue in that the cells can be precultured with various substances such as growth factors prior to grafting or they can be co-grafted with other cells or substances which promote specific parameters of differentiation. Furthermore, glial cells may have specific regional effects and produce neuronal growth factors (Barbin, G. et al., Devel. Neurosci. 7: 296-307 (1985); Schurch-Rathgeb, Y. et al., Nature 273: 308-309 (1978); Unsicker, K. et al. Proc. Natl. Acad. Sci. USA 81: 2242-2246 (1984); Whitaker-Azmitia, P. M. et al., Brain Res. 497: 80-85 (1989)). This suggests that co-transplanting cells providing the desired neurotransmitters along with specific types of glia that produce glial-derived factors, may promote neuronal growth and the desired differentiation of grafted cells.
Although early clinical experiments using the grafting approach did not result in long-lasting effects, an initial report of one study appeared more promising (Madrazo et al., Soc. Neurosci. Abstr. 12: 563 (1986); for an overview, see: Lieberman, A. et al., Adv. Tech. Stand. Neurosurg. 17: 65-76 (1990), which is hereby incorporated by reference). However, the surgical procedure used required craniotomy or full “open brain” surgery in which a portion of healthy striatum was removed and replaced with “chunks” of fetal adrenal gland. The therapeutic results obtained were somewhat controversial. However, both the need for serious neurosurgery in an already debilitated population and the need to use fetal tissue makes this approach undesirable.
Transdetermination has been observed in lower orders such as Drosophila, where a sample of cultured imaginal cells sometime differentiate into a structure appropriate to an imaginal disc other than that from which the culture was derived. Transdetermination represent a switch from one heritable state to another and so resembles the consequence of genetic mutation. (see Bruce Alberts, et al., Molecular Biology of the Cell, 1983, Garland Publishing Co., New York, N.Y., Ch. 15, pages 838-839). This phenomenon has been reported as occurring with groups of cells, but with cells of both the mutant and normal genotypes present.
Neural progenitor/stem cells obtained from fetal tissue or non-human tissue have been shown to be effective for cell replacement therapy for neurodegenerative disorders, head trauma, stroke and spinal cord injuries, and have been extrapolated to predict similar efficacy in repair of any type of nerve cell or brain cell damage. It has been recently found that neural progenitor/stem cells exist in the adult human brain, and that when these cells are cultured, the cells repeatedly divide and can (under the appropriate influences well defined in the art) differentiate into neurons, astorcytes and oligodendroglia.
In addition, PCT application WO 98/07841 discloses that one human cross species embryo was produced using human oral cavity epithelium as the donor nucleus. One NT unit developed to what was asserted to be a blastocyst stage embryo. This was placed on a feeder layer of cells. A cell mass appeared on the plate. However, there was no report of any information to suggest that the cell line was of human origin.
U.S. Pat. No. 6,087,168 (Levesque et al.) describes a method of converting, or transdifferentiating the epidermal cells into viable neurons useful in both cell therapy and gene therapy treatment methodologies. The method of transdifferentiating epidermal cells into neuronal cells comprises the following steps: obtaining skin cells from a patient; dedifferentiating these cells with an appropriate medium, neurotrophin or cytokine; transfecting the skin cells with one or more expression vector(s) encoding at least one neurogenic transcription factor or active fragments thereof, expressing at least one of the neurogenic transcription factors; growing the transfected cells in an appropriate medium; and adding to the medium one or more antisense oligonucleotide(s) corresponding to at least one negative regulator of neuronal differentiation, whereby the epidermal cells are transdifferentiated into neuronal cells. The Experimental Basis of that Invention is described as a transdifferentiation process involving the following basic steps of:
1. Isolation of proliferating epidermal basal cells from the skin of a patient in need;
2. Dedifferentiation of epidermal basal cells in calcium free growth media;
3. Expression of neurogenic basic-Helix-Loop-Helix (NeuroD1, NeuroD2, ASH1) and/or Zn-finger (Zic3, MyT1) transcription factors with simultaneous suppression of the expression of homeobox genes MSX1 and bHLH transcription factor HES1 in epidermal basal cells; and
4. Growing cells resulting from step 3 (cells which over-express neurogenic transcription factors and have suppressed expression of MXS1 and HES1) in the presence of low concentrations of all-trans retinoic acid and various neurotrophins, such as, BDNF, NGF, NT-3, and NT-4.
In the first step of that invention, epidermal or skin cells are obtained from a patient in need. These epidermal cells are obtained or isolated via any type of surgical procedure. Preferably, these isolated cells are epidermal basal cells obtained from the skin of a patient. However, epithelial, or any other type of basal cell or proliferating cell population, can be used for the conversion of these cells into neurons.
In the second step of that process, preferentially epidermal basal cells are dedifferentiated in a calcium free growth medium. This step involves treatment of the cells obtained in step one so that the cells lose the majority of differentiation specific gene expression to become dedifferentiated, that is, more primitive or developmentally less advanced. The dedifferentiation process is significant in that it allows for reprogramming of the neuronal development pathway. Since calcium ions are required to support development of keratinocytes (skin cells) from basal cells, removal of calcium results in dedifferentiation of basal cells. In other proliferating cell types, however, calcium may not be necessary to support development of any particular developmental pathway that is being deregulated. Other means to achieve the desired end of dedifferentiation involve treating the cells with specific growth factor or cytokines. Also, altering the specific gene expression pathway that is responsible for differentiation of epidermal cells by genetic manipulation may be used instead of eliminating calcium in the growth media. Moreover, elimination of calcium may not be required if other than proliferating epidermal basal cells are used.
In the third step, the process of that invention utilizes molecular manipulation techniques to alter the cell differentiation pathway of epidermal cells. This alteration is accomplished by allowing for the expression of neurogenic transcription factors, such as the basic-Helix-Loop-Helix factors, Neuro D1, Neuro D2, or ASH1, and/or zinc-finger transcription factors, such as Zic3 or MyT1, while simultaneously, or near simultaneously, suppressing the expression of genes responsible for suppression of the neuronal development pathway, such as the basic-Helix-Loop-Helix factor HES1 and/or the homeobox factor MSX1. In addition to these genes, any other set of neurogenic and anti-neurogenic genes can be manipulated so as to achieve the desired end of transdifferentiation of epidermal cells or other proliferating cell types. Manipulations that can be used in this step of the inventive process include the use of variety of gene transfer protocols, such as microinjection of expression constructs, and a variety of DNA transfection techniques (such as, lipofections, liposomes, coprecipitation techniques, and different carriers), and viruses. Also protein transfer methods can be used to transiently express neurogenic transcription factors in the proliferating dediffernentiated cells. Finally, in the fourth step of that invention, the transdifferentiated cells are preferably grown in the presence of a retinoid, such as all trans retinoic acid or vitamin A derivatives. In addition, neurotrophins or cytokines, such as BDNF, NGF, NT-3, NT-4, IL-6, can be used to obtain a substantial population of transdifferentiated neuronal cells. This step is optional in that it is not required for transdifferentiation. However, treatment with a retinoid and at least one neurotrophin increases the number of cells obtained.